Recent discoveries identifying the heart as a source of stem cells have opened up new prospects for autologous cellular cardiomyoplasty. Such cardiac stem cells (CSCs) have been isolated from rodent hearts and from human surgical specimens. We have made substantial progress in isolating and expanding these cells from percutaneously obtained endomyocardial biopsies. When grown in suspension culture, CSCs form self-organizing spherical clusters that display several features of differentiating cardiomyocytes. We now propose to continue functional characterization of CSCs and cardiospheres in vitro and in vivo. Understanding the electrophysiology of CSCs and cardiospheres may provide insights into events in cardiac development and repair and is vital to assessing the safety and efficacy of cell transplantation. This application seeks to elucidate the molecular and electrophysiologic phenotype at different stages of differentiation. Previous work using mesenchymal stem cells and bone marrow cells has demonstrated improvement of cardiac function without significant engraftment or differentiation of the injected cells into functional cardiac myocytes. Autologous cardiac stem cells have the highest potential for engraftment and differentiation into functional myoctes. We propose to compare engraftment and differentiation after injection of CSCs or cardiospheres in an animal model of myocardial infarction. Cardiospheres may, by virtue of their 3-dimensional architecture, improve cell engraftment, multiplication, survival and differentiation. Clinical trials of myoblasts, (an autologous cell type currently in clinical trials) have reported a high incidence of ventricular arrhythmias probably secondary to lack of electrical integration of myoblasts. Our preliminary data reveals that cardiac stem cells express Cx43 and are capable of electrical coupling with ventricular myocytes in co- culture. Here, we plan to evaluate electrical coupling and arrhythmogenic potential of cells in vitro and in vivo, using optical mapping, microelectrode recordings and 2-photon microscopy. Another important feature of stem cells is their ability to secrete factors that have a beneficial effect on cardiac function. Identification of secreted factors by stem cells may permit direct delivery of growth factors immediately after injury obviating the need for cell expansion. We will use proteomic techniques to identify secreted factors.Given the potential of clinical translation, this work opens up the possibility of revolutionizing the treatment of heart failure.